This invention relates to a process for determining the concentration of beta-adrenergic antagonists and their active metabolites in body fluids, antibodies useful in the assay and the method for obtaining the antibodies.
Presently, beta-adrenergic antagonists comprising the aryloxypropylamine series are widely used in the treatment of various cardiovascular diseases including angina pectoris, unstable angina, ventricular arrhythmia as well as primary and secondary forms of hypertension, thyrotoxicosis and migraine headaches. Examples of these drugs include propranolol, alprenolol hydrochloride, pindolol, tazolol, butoxamine hydrochloride and the like. Despite the widespread utility of these drugs, the maximal application in these clinical situations has been hampered by a relative inability to ascertain the end point of therapy. Propanolol, for example, is a drug with markedly variable bio-availibility and the desired therapeutic response bears little relationship to the dose administered. Furthermore, the drug has very few side effects, but its undesired side effects represent, in large part, an excess of the desired pharmacological response. To varying degrees, the same problem exists with the other forms of the aryloxypropylamine beta-adrenergic antagonists.
Physicians rely heavily upon documentation of a basal sinus bradycardia as a reflection of an adequate drug dose. However, this single clinical parameter dos not necessarily bear a predictable relationship to the adequate suppression of catecholamine effects with maximal exercise which is a better measure of sufficient beta-adrenergic blockade. This approach is more reliable and can be carried out in an exercise laboratory, but it is clearly an expensive, time-consuming approach which is not applicable to this serial evaluation of chronically ill patients.
Many prior attempts have been made to employ laboratory methods for the detection of propranolol in body fluids. These methods, which include fluorometric assay, radioreceptor assay and organic extraction with gas chromatography, share the features of relative insensitivity, excessive cost, or excessive time required for the processing of a single sample, making the approach unwieldy for large numbers of determinations or repeated determinations in a single patient. Furthermore, it has been documented that when the drug is orally administered, a significant fraction of the administered dose is converted by the liver to 4 OH-propranolol, a metabolite which retains full biological activity in its ability to bind to the beta-adrenergic receptor. High performance liquid chromatography, unlike many other methods, has been demonstrated to reliably measure this active metabolite. Nevertheless, high performance liquid chromatography is undesirable due to its excessive cost and time for the manipulation of blood samples. Naphthoxylactic acid is a second major metabolite of propranolol which, unlike 4 OH-propranolol, bears no biological activity. An additional drawback of several of the methods proposed for the measurement of propranolol in serum is the inability to distinguish this inactive metabolite from the native drug and the other active metabolites.
It has been proposed by Hoebeke et al, Biochemical Pharmacology, Vol. 27, pp. 1527-1532, (1978) to utilize a sensitive radioimmunoassay approach for propranolol and other beta-adrenergic blocking drugs. In this procedure, a specific antibody is raised in rabbits immunized with alprenolol hydrochloride (a cogenor of propanolol)covalently linked to bovine serum albumin. The covalent linkage is accomplished by reacting the olefin moiety of the alprenolol with N-bromosuccinimide to yield the corresponding bromohydrin. This bromohydrin then is reacted with blood serum albumin which has been reduced with dithiothreitol, to form the reactive sulfhydryl group. The covalent linkage is accomplished in an inert atmosphere in the dark by mixing the two reactants in solution at about 4.degree. C. for about 48 hours. The product then is purified such as by dialysis against deionized water. This conjugate in a phosphate buffered saline is administered intravenously to rabbits to raise the antibody which is recovered from the animals. The procedure is undesirable since the process of forming the bromide derivative is not high-yield. The process is also complicated by the fact that SH-groups must be substituted onto the protein carrier. Furthermore, the procedure is undesirable since the quantity of the antibody obtained from an animal is low so that the cost of immunization and subsequent recovery of antibody is undesirably expensive. These drawbacks outweigh the prime advantage of the Hoebeke et al process; namely, the antibody obtained is capable of determining the presence of other beta-adrenergic blocking drugs such as acebutolol, alprenolol, propranolol or other drugs which have the active propranolamine or ethanolamine side chain as well as active metabolites, but not the inactive metabolites.
It would be highly desirable to provide a means for assaying the concentration of beta-adrenergic antagonists as well as their active metabolites while avoiding the detection of inactive metabolites. Furthermore, it would be desirable to provide such an assay which utilizes an antibody that can be obtained from a given animal in much larger amounts than presently available antibodies. In addition, it would be desirable to provide a simplified procedure for raising such an antibody.